The two specific aims in this proposal address fundamental mechanisms of regulating chromatin structure for transcription that are relevant to cancer. The SWI/SNF nucleosome-remodeling complex is present from yeast to humans where it functions as a tumor suppressor. We have found that the Snf2 ATPase subunit of the yeast complex is modified by acetylation, which causes its dissociation from acetylated histones. This is consistent with a model whereby acetylation of Snf2 disengages the SWI/SNF complex from target genes. In the first aim we will test this model in vitro and in vivo and further explore the consequences of Snf2 acetylation on SWI/SNF activities. Biochemical experiments will examine the effects of acetylating Snf2 on interaction of the complex with transcription factors, its recruitment to target genes, its function as an ATPase and its ability to remodel and displace promoter nucleosomes. In vivo studies will analyze the effect of Snf2 acetylation on SWI/SNF recruitment and function on candidate target genes. We will also perform genome wide analysis of the effect of Snf2 acetylation on the localization of the complex and its effect on gene expression. These studies will reveal fundamental information regarding the regulation of SWI/SNF activity, which will empower understanding its function as a tumor suppressor in humans. Acetylation of histone H3 on lysine 56 (AcK56) is enriched in several cancers and represents a mark of newly synthesized histones. Its presence in chromatin is indicative of incorporation of new histones into chromatin. The Set2 histone methyltransferase has been reported to function as a tumor suppressor in human cells and we have found that deletion of SET2 in yeast leads to the enrichment of H3AcK56 in transcribed regions. Set2 functions as part of the Set2/Rpd3 pathway by which RNA polymerase II signals for histone deacetylation during transcription. The fact that H3AcK56 accumulates in transcribed sequences in set2 mutations suggests that new histones replace the original histones in the absence of the Set2/Rpd3 pathway. In the second aim we will analyze the functions of Set2/Rpd3S pathway components in facilitating the retention of original histones during the passage of RNA polymerase II and preventing the insertion of H3AcK56 into transcribed chromatin. In addition we will test the role of the ISWI class of nucleosome remodelers in reassembling chromatin behind RNA polymerase II as we have found that they co-immunoprecipitate with Set2-methylated nucleosomes. These studies will reveal fundamental information regarding the process of reassembling chromatin behind RNA polymerase II, which is required to preserve the fidelity of transcription initiation. 1